The detection of various target analytes or molecules is an important tool for a variety of applications including diagnostic medicine, molecular biology research and detection of contaminants or pathogens, to name a few. The detection methods differ according to the physical characteristics of each target molecule. For example, protein detection methods typically involve antibody-based immunoassays or mass spectrometry, whereas PCR-based assays may be more appropriate for nucleic acid detection.
The development of immunoassays and advances in methods of nucleic acid amplification have significantly advanced the art of the detection of biological analytes. In spite of these advances, nonspecific binding of the analyte to be detected and general assay noise has remained a problem that has limited the application and sensitivity of such assays.
Furthermore, while several methods for detecting different analytes have evolved, the ability to detect numerous target analytes simultaneously has proven difficult. Detection of multiple proteins, for example, has been limited to conventional electrophoresis assays or immunoassays. There has not been a significant multiplexed protein detection assay or method. Accordingly, there is a great need for analyte detection methods that are sensitive, specific, and readily amenable to multiplex detection.
Synthetic nucleic acid ligands, or aptamers, are versatile molecules useful in disease therapy and molecular medicine. Researchers have taken advantage of the relative ease with which RNA libraries can be generated and searched to create synthetic RNA-based molecules with novel functional properties. Aptamers are nucleic acid binding species that interact with high affinity and specificity to selected ligands. These molecules are generated through iterative cycles of selection and amplification known as in vitro selection or SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Ellington et al., Nature 346, 818-22 (1990); and Tuerk et al., Science 249, 505-10 (1990)). Aptamers have been selected to bind diverse targets such as dyes, proteins, peptides, aromatic small molecules, antibiotics, and other biomolecules (Hermann et al., Science 287, 820-5 (2000)). High-throughput methods and laboratory automation have been developed to generate aptamers in a rapid and parallel manner (Cox et al., Nucleic Acids Res 30, e108 (2002)). Researchers have demonstrated that aptamers can impart allosteric control properties onto other functional RNA molecules. Such allosteric control strategies have been employed to construct and select in vitro signaling aptamers, in vitro sensors, and in vitro allosterically controlled ribozymes (Jhaveri et al., Nat Biotechnol 18, 1293-7 (2000); Roth et al., Methods Mol Biol 252, 145-64 (2004); and Stojanovic et al., J Am Chem Soc 126, 9266-70 (2004)).
Aptamers present powerful tools for detecting analytes. However, there is a need to couple the capacity of aptamers to bind diverse target ligands with practical signal output methods that are typically associated with PCR, gel electrophoresis, or microarray assays.